The long term, overall objectives of the proposed research are to gain insight into the tumorigenic mechanism of transcription factor-related chromosomal translocations, and to provide detailed molecular information useful for diagnosis, monitoring of treatment and therapeutic design. The proposed studies focus on characterization of the structure and function of the fusion between EWS (the breakpoint site for chromosomal translocations in Ewing's sarcoma) and WT1 (the Wilms tumor suppressor gene) that results from the t(11;22) chromosomal translocation associated with desmoplastic small round cell tumor, a developmental tumor of young people. The specific aims are: 1) To further define the structure of the EWS-WT1 fusion gene and its chimeric RNA transcripts and protein products in desmoplastic small round cell tumors by PCR-based strategies for generation, mapping, subcloning and sequence analysis of genomic junction fragments and chimeric transcripts of the EWS-WT1 fusion in desmoplastic small round cell tumor. These studies will serve to define the chromosomal breakpoint region, quantitatively and qualitatively characterize the chimeric products, identify possible variant translocations, and assess the feasibility of molecular analysis for diagnosis and patient monitoring. 2) To characterize the transcription factor activity of the EWS-WT1 chimeric product by transactivation assays with chimeric- gene/expression-vector constructs, cotransfected with reporter genes under regulation of the EGRL promoter, a WTl responsive element. In addition, identification of endogenous EWS-WT1 target genes and quantitative analysis of their products will be carried out. 3) To investigate the tumorigenic activity of the EWS-WT1 chimera by cell transformation assays and generation of transgenic mice. EWS-WT1- expression vector constructs will be used to transfect transformation sensitive cell lines, alone and in cotransfection assays with cooperating oncogene constructs to fully evaluate the transforming potential of this chimeric molecule. Transgenic mice will be generated with EWS-WT1 constructs under control of ubiquitous and specific promoter systems for analysis of transformation potential in the setting of normal development and physiology. 4) To establish, optimize long term culture and characterize desmoplastic small round cell tumor cell lines and maintain tissue availability by tumor heterotransplants in immunodeficient mice. Ibis objective is crucial for continued investigation of the in vivo role of this unique gene fusion. These studies will further our understanding of the consequences and significance of transcription factor-related translocation events in this particular tumor, and should provide insights applicable to other tumor systems.